Learn Hardcore Japanese: kinou ha sigoto wo yasumimasita.

は here is the topic marker, so 昨日は means “as for yesterday…”
Time words like 昨日, 今日, 明日 can drop に when they act as adverbials.
Choosing は instead of simply omitting a particle also adds a slight contrastive nuance: “Yesterday (unlike other days), I took the day off.”

Why is 仕事 marked with を? Isn’t 休む intransitive?

In 仕事を休む, 休む is used transitively: “to take a break/absent oneself from [something].”
The thing you’re absent from (in this case “work”) gets を.

I thought 休む meant “to rest” and was intransitive. How can it take を?

Japanese 休む has two main senses:
• Intransitive “to rest” (木陰で休む).
• Transitive “to take a break/absent oneself from [work, school, etc.]” (会社を休む).
Both share the same spelling but differ in whether they take を.

What tense and politeness level is 休みました, and how would I say it informally?

休みました = polite past form of 休む (“took the day off”).
Casual/plain past is 休んだ, so you’d say 昨日は仕事を休んだ in informal speech.

Do I need to include 私は at the beginning?

No. Japanese often omits the subject when it’s obvious from context.
Adding 私は (私は昨日は仕事を休みました) is grammatically fine but usually redundant.

Can I reorder the sentence, for example put 昨日 at the end or add a comma?

Word order is relatively flexible. Common versions:
• 昨日、仕事を休みました
• 昨日は仕事を休みました
Placing 昨日 at the end (e.g. 仕事を休みました、昨日) is grammatically possible but sounds marked or poetic without special context.